Immobilized proteins and methods and uses thereof

ABSTRACT

The invention relates to the field of covalently attaching proteins to a substrate, particularly to methods of immobilizing proteins by posttranslationally modifying a cysteine residue of said protein through the addition of functional groups. The invention also relates to biological molecules used in such techniques, including proteins, and detection methods and kits that utilize such immobilized proteins, such as a microdevice or “protein chip”, a high-throughput screening device, and for the microscopy of proteins on a surface.

PRIORITY DATA

This application is a continuation of U.S. patent application Ser. No.13/482,512, filed May 29, 2012, which is a continuation of U.S. patentapplication Ser. No. 12/225,359 filed on Sep. 18, 2008, now issued asU.S. Pat. No. 8,188,241, which is the national phase filing under 35U.S.C. 371 of PCT Application No. PCT/US2007/007257, filed Mar. 22,2007, which claims the benefit of U.S. Provisional Patent ApplicationSer. No. 60/785,249, filed Mar. 22, 2006, each of which are incorporatedherein by reference.

GOVERNMENT INTEREST

This invention was made with government support under GM021328 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to the field of covalently immobilizingproteins on a substrate, particularly to methods of immobilizingproteins by posttranslationally modifying a cysteine residue of saidprotein through the addition of functional groups.

The present invention also relates to biological molecules used in suchtechniques, including proteins, and detection methods and kits thatutilize such immobilized proteins, such as a microdevice or “proteinchip”, a high-throughput screening device, and for the microscopy ofproteins on a surface.

BACKGROUND OF THE INVENTION

Proteins are immobilized to various surfaces for a myriad of purposes,such as in the production of protein “chips” or other analytical toolsuseful in studying protein-ligand and protein-protein interactions.Devices in which such proteins are immobilized covalently are generallymore robust than their non-covalent counterparts.

Typically covalent immobilization has been accomplished throughreactions between electrophilic reagents and exposed nucleophilichydroxyl, amino, carboxylate, and sulfhydryl moieties found in the sidechains of naturally occurring amino acids located on the surface ofproteins. This approach has two problems. It is difficult to selectamong the many nucleophiles on the surface of the protein targeted forimmobilization and it is difficult to distinguish between thosenucleophiles on the protein and those in other biological molecules thatmay be present in a complex mixture. Thus, it is difficult to target aspecific site in a specific protein for covalent attachment.

As such, there exists a need for structures and methods for covalentimmobilization of proteins that does not rely upon nucleophilic moietiesfound in the side chains of naturally occurring amino acids.

SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates tocovalently immobilizing proteins to a substrate.

In one embodiment, the invention is directed to a method of covalentlyimmobilizing a protein, comprising (a) posttranslationally modifying acysteine residue of said protein through the addition of functionalgroups, and (b) immobilizing said protein by ligation of said functionalgroups to a substrate; wherein said protein comprises one or more of asoluble protein or a solubilized protein.

In another embodiment, the invention is directed to a method ofcovalently immobilizing a protein, comprising (a) alkylating thesulfhydryl moiety in a C-terminal CaaX motif of a protein through acatalysis selected from the group consisting of catalysis of a farnesylanalog with protein farnesyltransferase and catalysis of ageranylgeranyl analog with protein geranylgeranyltransferase, resultingin the addition of a functional group, and (b) immobilizing thederivatized protein of (a) to a substrate comprising one or more of anazide-derivatized surface, an alkyne-derivatized surface and aphosphine-derivatized surface; wherein said protein comprises one ormore of a soluble protein and a solubilized protein.

In a further embodiment, the invention is directed to an isolatedprotein comprising a soluble protein having a non-native C-terminal CaaXmotif in which the cysteine has been posttranslationally modified,resulting in the addition of a functional group and wherein said proteinhas been immobilized to a substrate by ligating the functional group tosaid substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments. These embodimentsmay be better understood by reference to one or more of these drawingsin combination with the detailed description of specific embodimentspresented herein.

FIG. 1 depicts the synthesis scheme for farnesyl analog 10 AZDPPaccording to an embodiment of the invention.

FIG. 2 depicts the synthesis scheme for propargyl ether derivatives offarnesyl diphosphate according to an embodiment of the invention.

FIG. 3 depicts the modification scheme for Green Fluorescent Protein(GFP) and Glutathione S-transferase (GST) with CaaX recognition motifsinserted according to an embodiment of the invention.

FIG. 4 depicts the synthesis scheme for linker 4 according to anembodiment of the invention.

FIG. 5 depicts the synthesis scheme for linker 5 according to anembodiment of the invention.

FIG. 6 depicts the synthesis scheme for the production of Azide andhydroxyl protected gold nanoparticles according to an embodiment of theinvention.

FIG. 7 depicts the synthesis scheme for the production of Alkyne andhydroxyl protected gold nanoparticles according to an embodiment of theinvention.

FIG. 8 depicts the synthesis scheme for the production of Azideprotected gold nanoparticles coupled to dansyl-alkyne according to anembodiment of the invention.

FIG. 9 depicts analogs of farnesyl diphosphate according to anembodiment of the invention.

FIG. 10 depicts the synthesis scheme for benzylazide derivatives offarnesyl diphosphate according to an embodiment of the invention.

FIG. 11 depicts the synthesis scheme for prenyl azide derivatives offarnesyl diphosphate according to an embodiment of the invention.

FIG. 12 depicts the synthesis scheme for propargylamine etherderivatives of farnesyl diphosphate according to an embodiment of theinvention.

FIG. 13 depicts the synthesis scheme for propalamide ether derivativesof farnesyl diphosphate according to an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention may be understood more readily by reference to thefollowing detailed description of particular embodiments of theinvention.

Particular advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

Before the compositions and/or methods are disclosed and described, itis to be understood that this invention is not limited to specificcompositions or methods, as such may, of course, vary, unless it isotherwise indicated. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

DEFINITIONS

For the purposes of the invention, the following terms shall have thefollowing meanings:

Moreover, for the purposes of the invention, the term “a” or “an” entityrefers to one or more of that entity; for example, “a protein” refers toone or more of those elements or at least one element. As such, theterms “a” or “an”, “one or more” and “at least one” can be usedinterchangeably herein. It is also to be noted that the terms“comprising,” “including,” and “having” can be used interchangeably.Furthermore, an element or means “selected from the group consisting of”or “comprising one or more of” refers to one or more of the elements inthe list that follows, including mixtures (i.e. combinations) of two ormore of the elements.

For the purposes of the invention, ranges may be expressed herein asfrom “about” one particular value, and/or to “about” another particularvalue. When such a range is expressed, another embodiment includes fromthe one particular value and/or to the other particular value.Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

Reference will now be made in detail to particular embodiments of theinvention.

In a certain embodiment, the invention relates to posttranslationallymodifying a cysteine residue of a soluble or solubilized protein throughthe addition of functional groups, and immobilizing the protein byligation of the functional groups to a substrate. In one embodiment, thecysteine is in a C-terminal CaaX motif of a soluble or solubilizedprotein and the posttranslational modification includes alkylation ofthe sulfhydryl moiety by a farnesyl analog through catalysis with aprotein farnesyltransferase (PFTase) or alkylation by a analog throughcatalysis with a protein geranylgeranyltransferase (PGGTase), resultingin a modified cysteine residue in the protein. In another embodiment,the X of the CaaX motif is A, S, M, L or Q. In another embodiment, theC-terminal CaaX motif is not native to the protein

In another embodiment, the functional group added through prenylation isan azide or an alkyne. In another embodiment, the substrate to which theprotein is immobilized is a glass, a polymer, a gel or a metal surface.In a certain embodiment, a glass substrate is an azide-derivatized glasssurface or a phosphine-derivatized glass surface.

In another embodiment, the invention relates to isolated proteins havinga non-native C-terminal CaaX motif in which the cysteine has beenposttranslationally modified, resulting in the addition of a functionalgroup, wherein the protein has been immobilized to a substrate byligating the functional group to the substrate.

In one embodiment of the invention, a method for the covalentimmobilization of soluble proteins on a surface through an unnaturalamino acid created by posttranslationally modifying a cysteine residuewith functional groups, is described. In another embodiment the cysteineresidue is posttranslationally modified with functional groups suitablefor “click” chemistry or a Staudinger ligation. In another embodiment,protein farnesyltransferase (PFTase) or proteingeranylgeranyltransferase (PGGTase) catalyzes the alkylation of thesulfhydryl moiety in the cysteine located in C-terminal CaaX motifs, byanalogs of farnesyl diphosphate (FPP) or geranylgeranyl diphosphate(GGPP), as exemplified in FIG. 3. In a particular embodiment, the X ofthe CaaX motif is A, S, M, L or Q.

One embodiment of the invention includes farnesyl analogs. In anotherembodiment, farnesyl analogs have the general structure:

where Y represents the reactive group used to immobilize the modifiedprotein.

In another embodiment, a farnesyl analog includes an analog comprisingone or more of the analogs depicted in FIG. 9. Referring to FIG. 9farnesyl diphosphate is shown for reference only and analogs aredepicted as a base structure having an “R”, with R group substituentsshown thereafter.

In another embodiment, the surface to which a protein is immobilizedincludes silica, polymers, gels and metals. In another particularembodiment, a silicon surface includes silicon, glass slides, glassbeads, SiO₂, and silicon nitride; a polymer surface includes polymerbeads and polymer films; a gel includes agarose and acrylamide gels; anda metal includes surfaces, films, and particles of platinum, gold,silver, copper, zinc sulfide, cadmium selenide, zinc sulfide-cappedcadmium selenide, titanium dioxide, aluminum and aluminum oxide, opalfilms, and ceramics. In one embodiment, a glass surface includes anazide-derivatized glass surface or a phosphine-derivatized glasssurface.

In an embodiment of the invention, the method of immobilizing a proteinis for the immobilization of a protein in a microdevice or “proteinchip”, a high-throughput screening device, for the microscopy ofproteins on a surface, a sensor, a signaling device, a reactor forbiocatalysis, or a tag for a bioassay.

In another embodiment of the invention, a protein to be immobilizedincludes a or solubilized protein. In another embodiment, a method ofimmobilizing a protein includes a method in which the native fold of theprotein is preserved. Particular proteins to be immobilized include butare not limited to enzymes, antibodies, hormones, antigenic proteins,receptor proteins, RNA and DNA binding proteins, signaling proteins,drug binding proteins, and pathogenic proteins. In another embodiment,proteins to be immobilized include but are not limited to hormonechorionic gonadotrophin receptor (HCG receptor), luteinizing hormonereceptor, TEM-8 cell surface receptor, epidermal growth factor receptor,fibroblast growth factor receptor, insulin-like growth factor receptor,immunotoxins, and immunoconjugates.

The invention also includes isolated proteins having a non-nativeC-terminal CaaX motif in which the cysteine has been alkylated,resulting in the addition of a functional group where the protein hasbeen immobilized to a surface by ligating the functional group to thesurface.

EXAMPLES

It should be appreciated by those skilled in the art that the techniquesdisclosed in the examples which follow represent techniques discoveredby the inventors to function well in the practice of the invention, andthus can be considered to constitute particular modes for its practice.However, those of skill in the art should appreciate, in light of thepresent disclosure, that many changes can be made in the specificembodiments disclosed herein which will still obtain a like or similarresult without departing from the spirit and scope of the invention.

Example 1 Preparation of Modified Green Fluorescent Protein (GFP) with aPFTase Recognition Site

The gene for the GFPuv protein was subcloned into a pQE-30Xa expressionvector in order to append a His₆ sequence to the N-terminus and theC-terminal RTRCVIA PFTase recognition site. A 714 bp fragment frompGFPuv containing GFPuv was amplified by PCR. The forward primercontained 28 bases, 17 of which were complementary to the vectorsequence upstream of the 5′ end, FP-GFP:5′-CCGGTAGCATGCATGAGTAAAGGAGAAG-3′ (SEQ ID NO:1). The GFP start codon isindicated in bold, and a SphI site is underlined. The reverse primercontained 57 bases, 21 of which were complementary to the of the 3′ endof RP-GFP: 5′-GACGATAAGCTTTTAAGCAATAACGCACCTAGTTCGTTTGTAGAGCTCATCCATGCC-3′ (SEQ ID NO:2). The primer contained a HindIII site (underlined),and a C-terminal yeast RTRCVIA farnesylation motif (bold). The PCRproduct was purified on a 1% agarose gel and extracted from the gelusing the GFX™ PCR DNA Gel band purification kit, available fromAmersham Biosciences. The purified DNA and pQE-30Xa, available fromQiagen were separately digested with SphI and HindIII and purified byGFX™ DNA purification kit, available from Amersham Biosciences, toobtain the 740 bp GFP-CVIA fragment and the 3477 bp linear pQE-30Xavector, respectively. The two fragments were ligated using Quick T4 DNAligase, available from New England Biolabs, and transformed intoEpicurian coli XL1-Blue electrocompetent cells by electroporation. Cellswere selected for growth on ampicillin/tetracyclin. Restriction analysisand sequencing confirmed the desired construct. Sequence analysis ofpQE-GFP-CVIA indicated that the GFP-CVIA module was free of mutations.Ligation of the GFP-CVIA module into pQE-30Xa gave pQE-GFP-CVIA with GFPexpression under control of the T5 promoter. The recombinant proteincontained an N-terminal His₆ affinity tag and a C-terminal RTRCVIAPFTase recognition site.

Construct pQE-GFP-CVIA was transformed into chemically competentEpicurian coli M15(PREP4) cells, available from Qiagen. Starter cultures(10 mL of LB, 100 μg/mL ampicillin) were grown overnight at 37° C., withshaking. Three cultures, each containing 800 mL of LB (100 μg/mLampicillin), were inoculated with 8 mL of the overnight culture and weregrown at 37° C., with shaking at 240 rpm, until OD₆₀₀=0.6 when IPTG wasadded to 1 mM (final concentration), incubation was continued for 5 h,and cells were harvested by centrifugation and stored at −80° C. Cellpaste (9 g) was resuspended in 36 mL sonication buffer (50 mM sodiumphosphate, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol(BME), and 1 mM phenylmethanesulfonyl fluoride-PMSF). Cells were lysedby sonication, three periods of 30 s each. The sample was centrifugedfor 30 min at 18000 rpm in a Beckman J20 rotor. The supernatant wasmixed with 5 mL of Ni—NTA agarose resin, available from Qiagen) andshaken for 1 h at 4° C. GFP-CVIA was eluted from the resin with 250 mMimidazole. Fractions were screened by fluorescence and by SDS-PAGE.Pooled fractions were dialyzed against four changes of 50 mM Tris,pH=7.0, and 10 mM BME. Dialyzed enzyme was concentrated in a Centriprepcentrifugal filter (10,000 cutoff, Millipore) and stored at −80° C. in3:7 glycerol/water. A 1 L fermentation gave ˜4.5 g of wet cell paste and˜10 mg of GFP-CVIA that was >95% pure by SDS-PAGE.

Example 2 Preparation of Modified Glutathione S-Transferase (GST)Protein a with PFTase Recognition Site

The gene for the GST protein was subcloned into a pQE-30Xa in order toappend an N-terminal His₆ affinity tag and a C-terminal RTRCVIA PFTaserecognition site to the enzyme. A 660 bp fragment from pET-42bcontaining GST was amplified by PCR. The forward primer contained 33bases, 21 of which were complementary to the vector sequence upstream ofthe 5′GST. FP_GST: 5′-ATACATAAGCTTATGTCCCCTATACTAGGTTAT-3′ (SEQID NO:3).The GST start codon is indicated in bold, and a BamHI site isunderlined. The reverse primer contained 51 bases, 15 of which werecomplementary to the 3′end of GST RP-GST:5′-TGAACCAAGCTTTTAGGCTATAACACAGCGCGTACGATCCGATTTTGGAGG-3′ (SEQ ID NO:4).A HindIII site (underlined) and the C-terminal yeast farnesylation site(bold—enconding RTRCVIA) were introduced. The PCR product was purifiedon a 1% agarose gel and extracted using the GFX™ DNA purification kit.The purified DNA and pQE-30Xa, available from Qiagen, were separatelydigested with BamHI and HindIII and purified using a GFX™ DNApurification kit to obtain the 690 bp GST-CVIA fragment and the 3362 bplinear pQE-30Xa vector, respectively. The two fragments were ligatedusing Quick T4 DNA ligase, available from New England Biolabs, andtransformed into XL1-Blue electrocompetent cells by electroporation.Cells were selected for growth on ampicillin/tetracyclin. Restrictionanalysis and sequencing confirmed the desired construct. Sequenceanalysis of pQE-GST-CVIA indicated that the GST-CVIA module was free ofmutations. Ligation of the GST-CVIA module into pQE-30Xa yieldedpQE-GST-CVIA with GST under control of the T5 promoter.

Construct pQE-GST-CVIA was transformed into chemically competentEpicurian coli M15(PREP4) cells, available from Qiagen. Starter cultures(10 mL of LB, 100 μg/mL ampicillin) were grown overnight at 37° C., withshaking. Three cultures, each containing 800 mL of LB (100 μg/mLampicillin), were inoculated with 8 mL of the overnight culture and weregrown at 37° C., with shaking at 240 rpm, until OD₆₀₀=0.6 when IPTG wasadded to 1 mM (final concentration), incubation was continued for 5 h,and cells were harvested by centrifugation and stored at −80° C. Cellpaste (10 g) was resuspended in 50 mL of sonication buffer (50 mM sodiumphosphate, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol(BME), and 1 mM phenylmethanesulfonyl fluoride (PMSF). Cells were lysedby sonication, three periods of 30 s each. The sample was centrifugedfor 30 min at 18000 rpm in a Beckman J20 rotor. The supernatant wasmixed with 5 mL of Ni—NTA agarose resin, available from Qiagen, andshaken for 1 h at 4° C. The resin was loaded onto a column. The flowthrough was collected and the column was eluted with washing buffer(sonication buffer minus PMSF) until the absorbance at 280 nm decreasedto baseline. GST-CVIA was eluted with washing buffer containing 250 mMimidazole. Fractions were screened for GST activity using the GST.TagAssay Kit, available from Novagen, and by SDS-PAGE. Pooled fractionswere dialyzed four times against 15 mM Tris, pH 7.0, and 10 mM BME.Dialyzed enzyme was concentrated in a Centriprep centrifugal filter10,000 cutoff, available from Millipore, and glycerol was added to 30%.The sample was flash frozen in liquid nitrogen and stored at −80° C. Oneliter of cell culture produced ˜5 g of wet cell paste and ˜12 mg ofGST-CVIA that was at least 95% pure by SDS-PAGE.

Example 3 Preparation of Farnesyl Analogs

The general synthesis scheme for the preparation of compound10-Azido-Disphosphate (10 AZDPP) is set forth in FIG. 1.

The general synthesis scheme for the preparation of propargyl etherderivatives of farnesyl diphosphate is set forth in FIG. 2.

The general synthesis scheme for the preparation of benzylazidederivatives of farnesyl diphosphate is set forth in FIG. 10.

The general synthesis scheme for the preparation of prenyl azidederivatives of farnesyl diphosphate is set forth in FIG. 11.

The general synthesis scheme for the preparation of propargylaminederivatives of farnesyl diphosphate is set forth in FIG. 12.

The general synthesis scheme for the preparation of propalamidederivatives of farnesyl diphosphate is set forth in FIG. 13.

Example 4 In Vitro Farnesylation of GFP-CVIA and GST-CVIA

Purified GFP-CVIA or GST-CVIA (20 μl; 350 μM) and 1.8 mM of farnesyldiphosphate or an analog prepared as described herein (4 μL) were addedto 156 μL of 25 mM phosphate buffer, pH 7.0, containing 10 mM MgCl₂, and10 μM ZnSO₄. The sample was incubated at 30° C. for 10 min beforeaddition 10 μL of yeast PFTase (250 nM), obtained from pET42-RAM2/RAM1using the method of Harris et al. Biochemistry 2002, 41, 10554-10562.After 1 hour at 30° C. an additional 20 μL of GFP-CVIA or GST-CVIA and10 μL of PFTase were added and the incubation was continued for 1 h. Thesamples were concentrated using Centricon YM-10, available fromMillipore, and the concentration of total protein was determined byBradford analysis. A generalized reaction scheme is shown in FIG. 3.Farnesylation of GFP-CIVA with 10 AZDPP and PE10DPP result in productsreferred to as GFP-N₃ and GFP-C₂, respectively. Farnesylation ofGST-CIVA with 10 AZDPP and PE10DPP result in products referred to asGST-N₃ and GST-C₂, respectively. Farnesylation of GFP-CIVA and GST-CIVAwith farnesyl diphosphate result in products referred to as GFP-F andGST-F, respectively.

Example 5 Immobilization of Derivatized Proteins

A. Immobilization by “Click” Ligation

Amine-coated glass slides were treated with a solution ofN,N′-disuccinimidyl carbonate (DSC, 15 mM) and N,N-diisopropylethylamine(DIPEA, 15 mM) in DMF overnight at rt with shaking. The slides werewashed five times (5 min each) with DMF and dried under N₂. Thesuccinimidyl derivatized slides were treated with a 5:1 solution oflinker 4 and linker 5, the synthesis of which are set out in FIGS. 4 and5 respectively, (5 mM total concentration) and DIPEA (5 mM) in DMFovernight at rt. The slides were rinsed with EtOAc (5 times-5 min each),dried under N₂ and then capped by treatment with a solution ofethanolamine (50 mM) in DMF for 3 h at rt. The blocked slides wererinsed with EtOAc (5 times-5 min each) and dried under N₂.

One microliter of a solution of alkyne-derivatized proteins GFP-C₂ andGST-C₂ and Cu⁺ complex (1.7 mM final concentration, prepared by mixingequal volumes of tris-benzyltriazolylmethylamine (TBTA),² CuSO₄ and TCPE(20 mM each)), in 1:3 glycerol/water was spotted into wells on azidoslides masked with a silicon membrane. The slides were kept in a humidchamber at 4° C. for 3 h before Block It™ solution (4 μL) was added toeach well and then were returned to the chamber for 5 h. The slides werewashed with PBST (5 min each), appropriate fluorescently-taggedantibodies (4 μg/mL) were added to the wells (4 μL), and the plates wereincubated at 4° C. The slides were then washed with PBST (5 times-5 mineach) and scanned using a Typhoon 8600 Variable Mode Imager.

The concentrations of spotted GFP-C₂ and GST-C₂ varied from 1 to 20 μM.No fluorescent signal was seen when the antibodies were added to controlwells that did not contain GFP or GST proteins. In order to distinguishbetween specific and non-specific binding, GFP-F and GST-F bearing afarnesyl group instead of the reactive alkyne moiety were used ascontrols. Background fluorescence was seen for both of the farnesylatedproteins. The fluorescence signals for wells containing proteins withcovalently attached alkyne groups were easily detected and significantlyabove background. Over a range of 1-10 μM, the intensity of the signalincreased by ˜50% for each 2-fold increase in the concentration of thederivatized protein.

The antibodies could be removed by treatment with an acidic salinesolution (125 mM glycine, 500 mM NaCl, 2.5% Tween 20®, pH 2) for 2 h at80° C. Incubation of the stripped slides with anti-GFP for 16 h at 4°C., only gave signals corresponding to locations of immobilized GFP. Asecond cycle of stripping, followed by incubation with anti-GST onlygave signals at the locations of immobilized GST. Thus, GFP and GSTremain attached to the slides under conditions sufficiently stringent todisrupt interactions between the immobilized proteins and theirrespective antibodies.

In another experiment, GFP-C₂ (25-100 μM) was immobilized by the “click”procedure set forth above, and the slide was analyzed directly byphosphorimaging without conjugation with fluorescent antiGFP. Afluorescent signal was detected after the slide had been thoroughlywashed with PBST. The slide was then allowed to stand in phosphatebuffer (pH 7.0) at 4° C. for two days. After two days, the signal hadonly diminished by 22%. Thus, GFP retained its native fold during theimmobilization and subsequent storage in buffer.

B. Immobilization by Staudinger Ligation

Diphenylphosphinothiol was attached to amine-derivatized slidesaccording to the procedure set forth by Soellner et al. J. Am. Chem.Soc. 2003, 125, 11790-11791 to give a surface-bound phosphinothioester.

Azido-derivatized proteins GFP-N₃ and GST-N₃ in DMF/H₂O (50:1) wereadded to phosphinothioester slides by spotting 1 μL with into theappropriate well. The Staudinger ligation was allowed to proceed for 1.5h in an enclosed chamber saturated with DMF. Block It™ solution (4 μL)was added to each well and the slides were allowed to stand for 2.5 h.The slides were washed once with DMF and four times with PBST (5 mineach). Four microliters of a solution of the appropriate antibodies (4μg/mL) were added to the wells and the plates were incubated at 4° C.The slide was then washed with PBST (5 times-5 min each) and scannedwith a Typhoon 8600 Variable Mode Imager.

Visualization of GFP-N₃ and GST-N₃ by fluorescent antibodies yielded adetectable signal, indicating that the proteins were successfullyimmobilized, but control slides with GFP-F and GST-F had highbackgrounds. Lower backgrounds were achieved when the ligation wascarried out in 50:1 DMF/water.

In a separate experiment, GFP-N₃ was immobilized by the Staudingerligation as set forth above and the slides were stored in buffer andvisualized by direct imaging of the GFP fluorophore over a period of 6days. The fluorescence intensity of spots corresponding to immobilizedGFP-N₃ and the GFP-F control decreased during the first 4 days; however,the difference between the intensities for immobilized GFP and thecontrol remained constant. After 4 days signals for the control sampleswere reduced to background levels; whereas, the signal for covalentlybound GFP remained constant, indicating a stable immobilization of theprotein on the substrate.

Example 6 Immobilization of Proteins on a Gold Particle Substrate

Linker protected gold nanoparticles were generally produced as follows.Acetic acid (12.5% of the total final volume) was dissolved in MeOH(87.5% final total volume) in a Teflon vial. Linkers (75 mM total finalvolume concentration), produced as described herein, were added to theacetic acid MeOH solution and allowed to shake for 5 min. Hydrogentetrachloroaurate(III) trihydrate (gold source, 75 mM finalconcentration) was added to the linker solution and allowed to shake for10 min. Then NaBH₄ (750 mM) was added and the reaction was allowed tostir overnight. The linker protected gold nanoparticles were purifiedusing a 10K MW cutoff Slide-A-Lyzer® Dialysis Cassette and dialyzed in a1:1 (v:v) mixture of HPLC grade MeOH and nanopure water. Dialysissolution was changed three times. Purified particles were transferred toa clean Teflon vial and MeOH was concentrated on rotoevaporator and thewater was removed by lypothozation.

A. General Procedures for Click Chemistry Assay

Gold nanoparticles were re-suspended in MeOH to an approximateconcentration of 0.75 mM reactive linker. Fluorescent probe dissolved intert-butyl alcohol was added to 100 μL of nanoparticles stock for finalconcentration of 213 nM and 219 nM respectively. CuSO₄ was dissolved intert-butyl alcohol:water (1:1, v:v) followed by the addition of TBTA andsodium ascorbate to give a final concentration of 358 nM, 384 nM and 9mM, respectively. This freshly prepared solution was added to thereaction mixture and allowed to shake for ten days. Products werepurified using a 10K MW cutoff Slide-A-Lyzer® Dialysis Cassette anddialyzed in a 1:1 (v:v) mixture of HPLC grade MeOH and nanopure water.Dialysis solution was changed three times. Purified particles weretransferred to a clean Teflon vial and MeOH was concentrated onrotoevaporator and the water was removed by lypothozation.

B. Preparation of Azide and Hydroxyl Protected Gold Nanoparticles

Azide and hydroxyl protected gold nanoparticles were produced using thescheme generally set forth in FIG. 6. Thiols 13 and 9 were added tosolution a (HOAc (12.5% total volume) dissolved in MeOH for a finalreaction concentration of 0.75 mM and 6.75 mM, respectively. HAuCl₄.3H₂Owas added to give a final concentration of 75 mM. NaBH₄ was dissolved inMeOH immediately before addition to the reaction vial). The resultingparticles (22) were deposited onto 200 mesh, CU silicon Monoxide supportfilm (Ted Pella, Inc.).

C. Preparation of Alkyne and Hydroxyl Protected Gold Nanoparticles

Alkyne and hydroxyl protected gold nanoparticles were produced using thescheme generally set forth in FIG. 7. Thiols 13 and 9 were added tosolution a (HOAc (12.5% total volume) dissolved in MeOH for a finalreaction concentration of 0.38 mM and 6.75 mM, respectively. HAuCl₄.3H₂Owas added to give a final concentration of 75 mM. NaBH₄ was dissolved inMeOH immediately before addition to the reaction vial). The resultingparticles 23 were deposited onto 200 mesh, CU silicon monoxide supportfilm.

D. Preparation of Azide Protected Gold Nanoparticles Coupled toDansyl-Alkyne

Azide protected gold nanoparticles were coupled to dansyl-alkyne usingthe scheme generally set forth in FIG. 8 in a reaction containing CuSO₄,TBTA, and Sodium ascorbate, at room temperature, overnight.

The compositions and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of particular embodiments, it will be apparentto those of skill in the art that variations may be applied to thecompositions and methods and/or in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain related components may be substituted for thecomponents described herein while the same or similar results would beachieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

What is claimed is:
 1. A method of covalently immobilizing a protein,comprising: a. posttranslationally modifying a cysteine residue of saidprotein through the addition of functional groups, and b. immobilizingsaid protein by ligation of said functional groups to a substrate;wherein said protein comprises a soluble protein or a solubilizedprotein, and wherein said substrate comprises one or more of anazide-derivatized surface and an alkyne-derivatized surface.
 2. Themethod of claim 1, wherein said cysteine is in a C-terminal CaaX motifof a soluble protein.
 3. The method of claim 2, where X is A, S, M, L orQ.
 4. The method of claim 1, wherein said posttranslational modificationof a cysteine includes alkylation of the sulthydryl moiety comprisingone or more of catalysis of a farnesyl analog with proteinfarnesyltransferase and catalysis of a geranylgeranyl analog withprotein geranylgeranyltransferase.
 5. The method of claim 4, where saidfunctional group comprises one or more of an azide, an alkyne, analkene, a ketone and an alkoxyamine.
 6. The method of claim 4, whereinsaid farnesyl analog is selected from the analogs depicted in FIG.
 9. 7.The method of claim 1, wherein said functional group comprises one ormore of an azide and an alkyne.
 8. The method of claim 1, wherein saidsubstrate comprises one or more of a silicon surface selected from thegroup consisting of silicon, glass, SiO₂, and silicon nitride, a polymersurface comprising one or more of polymer beads and polymer films, a gelsurface comprising one or more of an agarose gel and an acrylamide gel,and a metal surface comprising one or more of platinum, gold, silver,copper, zinc sulfide, cadmium selenide, zinc sulfide-capped cadmiumselenide, titanium dioxide, aluminum, aluminum oxide, opal films, andceramics.
 9. The method of claim 8, wherein said substrate comprises oneor more of an azide-derivatized glass surface and an alkyne-derivatizedglass surface.
 10. A method of covalently immobilizing a protein,comprising: a. alkylating the sulthydryl moiety in a C-terminal CaaXmotif of a protein through a catalysis selected from the groupconsisting of catalysis of a farnesyl analog with proteinfarnesyltransferase and catalysis of a geranylgeranyl analog withprotein geranylgeranyltransferase, resulting in the addition of afunctional group, and b. immobilizing the derivatized protein of (a) toa substrate comprising one or more of an azide-derivatized surface andan alkyne-derivatized surface; wherein said protein comprises one ormore of a soluble protein and a solubilized protein.
 11. The method ofclaim 10, where X is A, S, M, L or Q.
 12. The method of claim 10, wheresaid functional group comprises one or more of an azide, an alkene, analkyne, a ketone, or an alkoxyamine.
 13. The method of claim 10, whereinsaid farnesyl analog is selected from the analogs depicted in FIG. 9.